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US8017319B2 - Evanescence-based multiplex sequencing method - Google Patents

Evanescence-based multiplex sequencing method Download PDF

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US8017319B2
US8017319B2 US10/499,547 US49954705A US8017319B2 US 8017319 B2 US8017319 B2 US 8017319B2 US 49954705 A US49954705 A US 49954705A US 8017319 B2 US8017319 B2 US 8017319B2
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nucleic acid
acid molecules
support
building blocks
immobilized
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US20070020626A1 (en
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Rudolf Rigler
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

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  • the invention concerns a method and a device for an evanescence-based multiplex sequencing of nucleic acid molecules immobilized on a support.
  • the sequencing of the human genome consisting of about 3 ⁇ 10 9 bases or of the genome of other organisms as well as the determination and comparison of individual sequence variants requires the provision of sequencing methods that are rapid and can also be used routinely and inexpensively.
  • major attempts have been made to accelerate conventional sequencing methods such as the enzymatic chain termination method of Sanger et al. (Proc. Natl. Acad. Sci. USA 74 (1977) 5463) especially by automation (Adams et al. Automated DNA Sequencing and Analysis (1994), New York, Academic Press), at present no more than 2000 bases per day can be determined with a sequencer.
  • Another method is single molecule sequencing (Dörre et al., Bioimaging 5 (1997), 139-152) in which nucleic acids are sequenced by progressive enzymatic degradation of fluorescent-labelled single-stranded DNA molecules and detection of the sequentially released monomer molecules in a microstructure channel.
  • the advantage of this method is that only a single molecule of the target nucleic acid is sufficient to carry out a sequence determination.
  • the object of the present invention was to provide a method for sequencing nucleic acids which represents a further improvement over the prior art and which allows a parallel determination of individual nucleic acid molecules in a multiplex format.
  • a multiplex sequencing method is proposed in PCT/EP01/07462 in which nucleic acid molecules that carry several fluorescent marker groups are provided in an immobilized form on a support and the base sequence of several nucleic acid molecules is determined simultaneously on the basis of the time-dependent change in the fluorescence of the nucleic acid molecules or/and of the cleaved nucleotide building blocks caused by the cleavage of nucleotide building blocks.
  • the subject matter of the present application is a method for sequencing nucleic acids comprising the steps:
  • Yet another subject matter of the invention is a device for sequencing nucleic acids comprising:
  • the method according to the invention and the device according to the invention can be used for example to analyse genomes and transcriptomes or for differential analyses e.g. investigations of differences in the genome or transcriptome of individual species or organisms within a species.
  • FIG. 1 is a schematic view of an optically transparent support, according to the invention.
  • FIG. 2 is a schematic view showing excitation light beamed into the optically transparent support of FIG. 1 ;
  • FIG. 3A is a schematic view showing evanescent excitation fields generated by multiple reflections in the optically transparent support of FIG. 1 ;
  • FIG. 3B is a schematic view showing evanescent excitation fields in the form of strips.
  • FIG. 3C is a schematic view showing evanescent excitation fields in the form of points.
  • the method according to the invention is a support-based multiplex sequencing method in which a multitude of immobilized nucleic acid molecules are examined simultaneously.
  • the support used for the method can be any desired planar or structured support that is suitable for immobilizing nucleic acid molecules and has, at least in the area of the immobilized nucleic acids sufficient optical transparency and suitable surface properties for an evanescence-based detection of fluorescence.
  • suitable support materials are glass, quartz, plastic or composite materials containing these materials.
  • the support can be designed in any manner, provided a reaction space can be formed which allows the progressive cleavage of individual nucleotide building blocks from nucleic acids immobilized on the support in a liquid reaction mixture.
  • the nucleic acid molecules that can be in a single-stranded form or in a double stranded form are preferably immobilized on the support via their 5′ or 3′ ends. In the case of double-stranded molecules it must be ensured that labelled nucleotide building blocks can only be cleaved from a single strand.
  • the nucleic acid molecules can be bound to the support by means of covalent or non-covalent interactions.
  • the binding of polynucleotides to the support can be mediated by high affinity interactions between the partners of a specific binding pair, e.g. for example mediated by biotin/streptavidin or avidin, hapten/anti-hapten-antibody, sugar/lectin etc.
  • biotinylated nucleic acid molecules can be coupled to streptavidin-coated supports.
  • the nucleic acid molecules can also be adsorptively bound to the support.
  • nucleic acid molecules modified by incorporation of alkanethiol groups can be bound to metallic supports e.g. gold supports.
  • Another alternative is covalent immobilization in which the binding of the polynucleotides can be mediated by reactive silane groups on a silica surface.
  • a plurality of nucleic acid molecules that are to be sequenced are bound to a support.
  • the bound nucleic acid fragments have a length of preferably 200 to 2,000 nucleotides, particularly preferably 400 to 1,000 nucleotides.
  • the nucleic acid molecules bound to the support e.g. DNA molecules or RNA molecules, contain a plurality of fluorescent marker groups and preferably at least 50%, particularly preferably at least 70% and most preferably essentially all, e.g. at least 90%, of the nucleotide building blocks of one base type carry a fluorescent marker group.
  • Nucleic acids labelled in this manner can be produced by enzymatic primer extension on a nucleic acid template using a suitable polymerase e.g. a DNA polymerase such as Taq polymerase, a thermostable DNA polymerase from Thermococcus gorgonarius or other thermostable organisms (Hopfner et al., PNAS USA 96 (1999), 3600-3605) or a mutated Taq polymerase (Patel and Loeb, PNAS USA 97 (2000), 5095-5100) using fluorescent-labelled nucleotide building blocks.
  • a suitable polymerase e.g. a DNA polymerase such as Taq polymerase, a thermostable DNA polymerase from Thermococcus gorgonarius or other thermostable organisms (Hopfner et al., PNAS USA 96 (1999), 3600-3605) or a mutated Taq polymerase (Patel and Loeb, PNAS USA 97
  • the labelled nucleic acid molecules can also be produced by amplification reactions e.g. PCR.
  • amplification reactions e.g. PCR.
  • amplification products are formed in which only a single strand contains fluorescent labels.
  • Such asymmetric amplification products can be sequenced in a double-stranded form.
  • Nucleic acid fragments are formed by symmetrical PCR in which both strands are fluorescent labelled.
  • These two fluorescent labelled strands can be separated and immobilized separately in a single-stranded form so that the sequence of one or both complementary strands can be determined separately.
  • one of the two strands can be modified at the 3′ end for example by incorporation of a PNA clip, such that monomer building blocks can no longer be cleaved off. In this case double-strand sequencing is possible.
  • nucleotide building blocks of at least two base types for example two, three of four base types, carry a fluorescent label and each base type advantageously carries a different fluorescent marker group. If the nucleic acid molecules are not completely labelled, the sequence can nevertheless be completely determined by sequencing a plurality of molecules in parallel.
  • the nucleic acid template whose sequence is to be determined can for example be selected from DNA templates such as genomic DNA fragments, cDNA molecules, plasmids etc. and also from RNA templates such as mRNA molecules.
  • the fluorescent marker groups can be selected from known fluorescent marker groups used to label biopolymers e.g. nucleic acids such as fluorescein, rhodamine, phycoerythrin, Cy3, Cy5 or derivatives thereof etc.
  • the method according to the invention is based on the fact that fluorescent marker groups incorporated into nucleic acid strands interact with neighbouring groups, for example with chemical groups of the nucleic acids and in particular with nucleobases such as G, or/and with adjacent fluorescent marker groups which results in a change in the fluorescence and in particular in the fluorescence intensity compared to that of the fluorescent marker groups in an isolated form due to quenching or/and energy transfer processes.
  • Cleavage of individual nucleotide building blocks results in a change in the total fluorescence e.g. the fluorescence intensity of an immobilized nucleic acid strand is changed in a manner depending on the cleavage of individual nucleotide building blocks i.e. as a function of time.
  • This change in the fluorescence over time can be detected in parallel for a plurality of nucleic acid molecules and can be correlated with the base sequence of individual nucleic acid strands.
  • Fluorescent marker groups are preferably used which are at least partially quenched when they are incorporated into the nucleic acid strand such that after cleavage of the nucleotide building block containing the marker group or of a neighbouring building block which causes the quenching, the fluorescence intensity is increased.
  • the sequencing reaction of the method according to the invention comprises the progressive cleavage of individual nucleotide building blocks from the immobilized nucleic acid molecules.
  • An enzymatic cleavage is preferably carried out using an exonuclease in which single strand or double strand exonucleases that degrade in the 5′ ⁇ 3′ direction or 3′ ⁇ 5′ direction can be used depending on the manner in which the nucleic acid strands are immobilized on the support.
  • T7 DNA polymerase, E. coli exonuclease I or E. coli exonuclease III are particularly preferably used as exonucleases.
  • a change in the fluorescence intensity of the immobilized nucleic acid strand or/and of the cleaved nucleotide building blocks due to quenching or energy transfer processes can be measured during the progressive cleavage of individual nucleotide building blocks.
  • This change in the fluorescence intensity over time depends on the base sequence of the examined nucleic acid strand and can therefore be correlated with the sequence.
  • several nucleic acid strands labelled on different bases e.g.
  • A, G, C and T or combinations of two different bases are preferably generated by enzymatic primer extension as described above and immobilized on the support where the immobilization can be at random sites on the support or can be carried out in a site-specific manner.
  • a sequence identifier may optionally also be attached to the nucleic acid strand to be examined e.g. a labelled nucleic acid of a known sequence, for example by means of an enzymatic reaction using ligase or/and terminal transferase such that firstly a known fluorescence pattern is obtained at the start of the sequencing and the fluorescence pattern of the unknown sequence to be examined is only obtained afterwards.
  • a total of 10 3 to 10 6 nucleic acid strands are immobilized on a support.
  • a convection flow is preferably generated in the reaction space away from the support.
  • the flow rate can be in the range of 1 to 10 mm/s.
  • the detection comprises beaming light into the support preferably by means of a laser.
  • One or several laser beams can be used for this e.g. a widened laser beam with a cross-section of ca. 1-20 mm or/and multiple laser beams.
  • An evanescent excitation field is generated by internal reflection at one or more positions of the support surface in the area of immobilized nucleic acid molecules which excites the fluorescent marker groups on the nucleic acid molecules immobilized on the support.
  • the reflection on the support surface is preferably a total internal reflection.
  • the fluorescence emission of a plurality of nucleic acid strands generated by evanescent excitation can be detected in parallel using a detector matrix which for example comprises an electronic detection matrix e.g. a CCD camera or an avalanche photodiode matrix. Detection can be such that fluorescence excitation and detection occurs concurrently on all examined nucleic acid strands. Alternatively the nucleic acid strands can be examined portion by portion in several steps. It is preferable to detect the fluorescence light that is irradiated essentially orthogonally from the support surface.
  • a detector matrix which for example comprises an electronic detection matrix e.g. a CCD camera or an avalanche photodiode matrix. Detection can be such that fluorescence excitation and detection occurs concurrently on all examined nucleic acid strands. Alternatively the nucleic acid strands can be examined portion by portion in several steps. It is preferable to detect the fluorescence light that is irradiated essentially orthogonally from
  • FIG. 1 shows a schematic representation of an optically transparent support ( 2 ) according to the invention with a multitude of single-stranded labelled nucleic acid molecules ( 4 ) immobilized thereon.
  • a support with an area of 1 to 2 cm 2 can for example contain up to 10 6 nucleic acid strands.
  • FIG. 2 shows a first embodiment of the invention in which excitation light ( 6 ) is beamed into the optically transparent support ( 2 ) with nucleic acid molecules ( 4 ) immobilized thereon by a widened laser and the light emerges again from the support ( 2 ) after reflection at the glass surface in the area of the immobilized nucleic acid molecules ( 4 ).
  • the immobilized nucleic acid molecules ( 4 ) are excited to fluoresce by the evanescent excitation field.
  • the emission light ( 8 ) is guided by an optical system ( 10 ) onto a detector ( 12 ).
  • evanescent excitation fields are generated by multiple reflections ( 14 a , 14 b , 14 c ) in the optically transparent support ( 2 ).
  • the evanescent excitation fields can for example be present in the form of strips ( FIG. 3B ) or points ( FIG. 3C ).

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US10/499,547 2001-12-19 2002-12-18 Evanescence-based multiplex sequencing method Expired - Lifetime US8017319B2 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE10162536A DE10162536A1 (de) 2001-12-19 2001-12-19 Evaneszenz-basierendes Multiplex-Sequenzierungsverfahren
DE10162536.7 2001-12-19
DE10162536 2001-12-19
PCT/EP2002/014490 WO2003052137A2 (fr) 2001-12-19 2002-12-18 Procede de sequençage multiplex base sur le principe de l'evanescence

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US20070020626A1 US20070020626A1 (en) 2007-01-25
US8017319B2 true US8017319B2 (en) 2011-09-13

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US (1) US8017319B2 (fr)
EP (1) EP1458892B1 (fr)
AT (1) ATE518963T1 (fr)
AU (1) AU2002361162A1 (fr)
DE (1) DE10162536A1 (fr)
WO (1) WO2003052137A2 (fr)

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102004038359A1 (de) 2004-08-06 2006-03-16 Rudolf Rigler Paralleles Hochdurchsatz-Einzelmolekül-Sequenzierungsverfahren
CN102653784B (zh) * 2011-03-03 2015-01-21 深圳华大基因科技服务有限公司 用于多重核酸测序的标签及其使用方法
WO2013131888A1 (fr) * 2012-03-06 2013-09-12 Rudolf Rigler Procédé de séquençage de molécule cyclique unique
US11091805B2 (en) 2014-01-10 2021-08-17 Gnothis Holding Ag Single molecule analysis with high accuracy
EP3112841A1 (fr) 2015-06-30 2017-01-04 Gnothis Holding SA Analyse de molécule unique dans un champ électrique
US20230365612A1 (en) * 2022-04-29 2023-11-16 454 Corporation Compound including a heteroatom and nucleobase
EP4379378A1 (fr) 2022-11-29 2024-06-05 Gnothis Holding AG Surface résistante aux protéines revêtue sur une zone sélective
WO2024099959A1 (fr) 2022-11-07 2024-05-16 Gnothis Holding Ag Surface résistante aux protéines revêtue d'une zone sélective
EP4403643A1 (fr) 2023-01-19 2024-07-24 Gnothis Holding AG Sites de liaison moleculaire multiples
EP4623095A1 (fr) 2022-11-21 2025-10-01 Gnothis Holding AG Sites de liaison moléculaires multiples
EP4375644A1 (fr) 2022-11-22 2024-05-29 Gnothis Holding AG Analyse d'empreintes digitales d'événements moléculaires uniques
EP4379355A1 (fr) 2022-11-29 2024-06-05 Gnothis Holding AG Analyse à haut débit d'événements de molécule unique
EP4382614A1 (fr) 2022-12-06 2024-06-12 Gnothis Holding AG Support à haut rendement

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WO2003052137A3 (fr) 2004-01-15
EP1458892A2 (fr) 2004-09-22
DE10162536A1 (de) 2003-07-17
WO2003052137A2 (fr) 2003-06-26
AU2002361162A8 (en) 2003-06-30
US20070020626A1 (en) 2007-01-25
AU2002361162A1 (en) 2003-06-30
ATE518963T1 (de) 2011-08-15
EP1458892B1 (fr) 2011-08-03

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